Application of pseudomonas aeruginosa vaccine in respiratory disease

ABSTRACT

The present invention provides use of a  Pseudomonas aeruginosa  vaccine in the manufacture of a medicament for the prevention and treatment of respiratory system disease. The  Pseudomonas aeruginosa  vaccine of the present invention can effectively prevent and treat pulmonary infection caused by multidrug-resistant  Pseudomonas aeruginosa  and COPD complicated with  Pseudomonas aeruginosa  infection by activating the specific immune response of the body. The  Pseudomonas aeruginosa  vaccine of the present invention can reduce the bacterial load in the immunized subject through the established immunization procedures, thereby providing a technical solution that can effectively prevent pulmonary infection with  Pseudomonas aeruginosa,  which avoids the technical problems caused by the use of antibiotics such as poor effectiveness, difficulty in curing and proneness to drug resistance in the prior art to a certain degree.

PRIORITY APPLICATIONS

The present application claims priority from Chinese invention patent applications 1) 201910777479.2 “BACTERIAL MEMBRANE VESICLE, AND PREPARATION METHOD AND APPLICATION THEREOF”, 2) 201910777473.5 “STAPHYLOCOCCUS AUREUS MEMBRANE VESICLE, AND PREPARATION METHOD AND APPLICATION THEREOF”, 3) 201910777606.9 “PSEUDOMONAS AERUGINOSA MEMBRANE VESICLE, AND PREPARATION METHOD AND APPLICATION THEREOF”, 4) 201921369450.2 “A PRODUCTION SYSTEM, AND ISOLATION AND PURIFICATION STSTEM FOR BACTERIAL MEMBRANE VESICLE”, 5) 201910777595.4 “A PRODUCTION SYSTEM, AND ISOLATION AND PURIFICATION STSTEM AND METHOD FOR BACTERIAL MEMBRANE VESICLE” filed on Aug. 22, 2019, which are incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention belongs to the field of microbiology, and particularly relates to an application of a pseudomonas aeruginosa vaccine in the field of respiratory system disease.

BACKGROUND

Chronic obstructive pulmonary disease, which is abbreviated as COPD and often referred to as chronic obstructive lung, is an obstructive pulmonary disease characterized by persistent airflow limitation. COPD is a progressive disease, that is, the medical condition becomes progressively worse over time. The main symptoms are shortness of breath, cough, and expectoration. COPD has complex causes and is mainly caused by cigarettes, air pollution and genetics, according to clinical observations. Smoking is the main cause. COPD has become a major problem which affects national health in the 21st century. According to statistics, about 329 million patients worldwide suffer from the disease every year, and COPD is currently the fourth leading cause of death in the world. WHO predicts that COPD will become the third leading cause of death in the world by 2030. So far, the pathogenesis of COPD has not been fully clarified. The existing pathogenesis includes the following hypotheses: “infection hypothesis”, “oxidative-antioxidative imbalance hypothesis” and “elastin-anti-elastin hypothesis”, as well as “chronic chlamydia pneumoniae infection hypothesis”, “immune imbalance hypothesis” and “autonomic nerve dysfunction” which have been put forward successively in recent years. However, these theories cannot fully explain all manifestations of all COPD patients.

Acute exacerbation of COPD (AECOPD) refers to the worsening of respiratory distress symptoms in patients, manifested as cough, expectoration, dyspnea worse than usual, or increased amounts of sputum, yellow sputum, or a need to change the medication regimen. AECOPD is an important cause of acute hospitalization of COPD patients, accounting for 2.4% of the total hospitalization rate. According to statistics, the mortality rate of first-time hospitalization patients within 7 days is about 11.6%; and the mortality rate of second-time hospitalization patients within 7 days is as high as 37%. The mortality rate of hospitalization patients within 30 days is about 15%. Common complications of AECOPD patients include: respiratory failure, chronic pulmonary heart disease and spontaneous pneumothorax. Studies prove that infection is an important factor in AECOPD, accounting for the first place by about 80%. A systematic retrospective analysis indicates that complicated bacterial infection accounts for the first place in the infection, about 50%, and the bacterial load of the lower respiratory tract is closely related to the prognosis. Viral infection accounts for the second place, about 43%.

Pseudomonas aeruginosa is a common opportunistic pathogen which can occur in any part and tissue of a human body, such as respiratory tract, middle ear, cornea and urethra, and is also common in burned or wounded sites. Pseudomonas aeruginosa often colonizes the lower respiratory tract of patients with moderate and severe COPD, and is relevant to the exacerbation of COPD. Existing studies show that the probability of COPD complicated with Pseudomonas aeruginosa infection is 15.5%, and the probability of patients with acute exacerbation of COPD complicated with Pseudomonas aeruginosa infection is 13%. The probability of patients with recurrent COPD complicated Pseudomonas aeruginosa infection is 23.1%.

Currently, Pseudomonas aeruginosa is the most common nosocomial pathogen in the United States and the second most common pathogen of ventilator-associated pneumonia. A previous study has indicated that 10.2% of AECOPD patients are rehospitalized within 30 days after discharge, and 17.8% are rehospitalized within 90 days, and the detection rate of Pseudomonas aeruginosa is positively correlated with the hospitalization rate of patients.

Pseudomonas aeruginosa in the pulmonary infection is relatively difficult to treat. Firstly, Pseudomonas aeruginosa has a complex drug resistance mechanism. At present, the detection rate of multidrug-resistant and extensively drug-resistant Pseudomonas aeruginosa is high. Secondly, Pseudomonas aeruginosa has a large genome (5.5-7Mbp) and many phenotypes, and can adapt to different colonization environments with strong mutation ability. Moreover, the bacteria can produce a variety of secondary metabolites and polymers, and have a stronger quorum sensing ability, which enables the acquired resistance to be transferred among bacteria. Thirdly, Pseudomonas aeruginosa secretes virulence factors which damage the immune system and are difficult to eliminate. As a result, patients use antibiotics for longer time and hospitalize for longer time, thereby often leading to secondary infections during hospitalization and antibiotic side effects.

The most common cause of death in patients with cystic fibrosis is the infection of Pseudomonas aeruginosa. The patient suffers from the recurrence of infection, inflammation, and airway obstruction, which will continuously damage the small airways. Aerosol inhalation of antibiotics is the most common treatment and is the most direct method for high-dose antibiotics to reach the site of infection. This treatment often cannot eradicate Pseudomonas aeruginosa infection even if clinical microbiology proves that pathogens are sensitive to the high-dose antibiotics. Pseudomonas aeruginosa forms antibiotic-resistant biofilms in the lungs of the patients with cystic fibrosis, which may be one of the reasons for the difficulty of antibiotic therapy.

SUMMARY

In view of the above technical problems, the present invention provides use of a Pseudomonas aeruginosa vaccine in the manufacture of a medicament for the prevention and treatment of respiratory system disease.

Further, the respiratory system disease comprises primary respiratory system disease and secondary respiratory system disease.

Further, the respiratory system disease is chronic pulmonary insufficiency complicated with bacterial infection.

Further, the chronic obstructive pulmonary insufficiency is chronic obstructive pulmonary disease.

Further, the chronic obstructive pulmonary disease comprises chronic bronchitis and emphysema.

Further, the chronic obstructive pulmonary disease is acute exacerbation of chronic obstructive pulmonary disease.

Further, the bacterial infection is caused by one or more of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae and Mycobacterium tuberculosis.

Further, the bacterial infection is Pseudomonas aeruginosa infection.

Further, immunization procedure of the Pseudomonas aeruginosa vaccine comprises: injection take places at (i) 0, 3rd, and 7th days; (ii) 0, 1st, and 2nd weeks; (iii) 0, 2nd, and 4th weeks; and (iv) 1st, 2nd, 3rd, and 4th weeks.

Further, the Pseudomonas aeruginosa vaccine comprises inactivated Pseudomonas aeruginosa and/or Pseudomonas aeruginosa membrane vesicles.

Further, the inactivated Pseudomonas aeruginosa is inactivated by irradiation, and the Pseudomonas aeruginosa membrane vesicles are isolated from the Pseudomonas aeruginosa inactivated by irradiation.

Further, the Pseudomonas aeruginosa vaccine prevents Pseudomonas aeruginosa infection, and reduces bacterial load in the respiratory system disease.

Further, content of whole-cell Pseudomonas aeruginosa in the Pseudomonas aeruginosa vaccine comprises: 1×10⁴-1×10¹¹/injection.

Further, the content of the whole-cell Pseudomonas aeruginosa in the Pseudomonas aeruginosa vaccine comprises: 1×10⁴/injection, 1×10⁵/injection, 1×10⁶/injection, 1×10⁷/injection, 1×10⁸/injection, 1×10⁹/injection and 1×10¹¹/injection.

Further, the Pseudomonas aeruginosa vaccine further contains an immunoadjuvant.

Further, the immunoadjuvant is aluminum hydroxide.

Further, administration site of the Pseudomonas aeruginosa vaccine is subcutaneous, muscle and/or mucosa.

Further, the medicament can also contain any pharmaceutically acceptable carrier and/or adjuvant.

Further, the carrier is a liposome.

The present invention also provides a pharmaceutical composition for treating respiratory system disease, wherein comprising a Pseudomonas aeruginosa vaccine and at least one antibiotic.

Further, the at least one antibiotic is aztreonam.

Further, the Pseudomonas aeruginosa vaccine comprises inactivated Pseudomonas aeruginosa and/or Pseudomonas aeruginosa membrane vesicles.

The Present Invention has the Following Beneficial Effects:

The experimental results of the present invention show that the Pseudomonas aeruginosa vaccine of the present invention can effectively prevent and treat pulmonary infection caused by multidrug-resistant Pseudomonas aeruginosa and COPD complicated with Pseudomonas aeruginosa infection by activating the specific immune response of the body. The Pseudomonas aeruginosa vaccine of the present invention can reduce the bacterial load in the immunized subject through the established immunization procedures, thereby providing a technical solution that can effectively prevent pulmonary infection with Pseudomonas aeruginosa , which avoids the technical problems caused by the use of antibiotics such as poor effectiveness, difficulty in curing and proneness to drug resistance in the prior art to a certain degree.

The experimental results of the present invention also show that, in one aspect, the Pseudomonas aeruginosa vaccine of the present invention avoids the problem of drug resistance of Pseudomonas aeruginosa from the principle of action through the stimulated specific immune protection, and may achieve a faster effect for eliminating pathogenic bacteria than antibiotics. In another aspect, the Pseudomonas aeruginosa vaccine of the present invention can also be used in combination with antibiotics (such as aztreonam) to produce a synergistic effect against infection. Therefore, the Pseudomonas aeruginosa vaccine of the present invention has broad application scenarios in the field of the respiratory system disease.

DESCRIPTION OF DRAWINGS

To make the embodiments of the present invention or the technical solutions in the prior art clearer, the drawings required to be used in the description of the embodiments or the prior art will be briefly introduced below. It is obvious that the drawings described below are some embodiments of the present invention, and that other drawings can be obtained from these drawings for those of ordinary skill in the art without making inventive effort.

FIG. 1 is a Transmission Electron Microscopy (TEM) image of irradiated Pseudomonas aeruginosa membrane vesicles (scale: 200 nm).

FIG. 2 shows the percentage of proliferation of CD4⁺T cells after interacting with DC treated with different treatment methods.

FIG. 3 is a flow cytometry plot of proliferation of CD4⁺T cells after interacting with DC treated with different treatment methods.

FIG. 4 shows irradiated membrane vesicles enhance the interaction between DC cells and T cells (GC: growth control, dendritic cell growth control group (unstimulated group); Cell+MVs (whole-cell bacteria+membrane vesicle treatment group); MVs (membrane vesicle treatment group)).

FIG. 5 shows the lung tissue of mice in the normal control group (left: ×40 magnification; right: ×200 magnification).

FIG. 6 shows the lung tissue of mice in the COPD model group (left: ×40 magnification; right: ×200 magnification).

FIG. 7 shows the bacterial load in the lung tissue of COPD mice 24h after Pseudomonas aeruginosa SKLBPA1 infection.

FIG. 8 shows immunoprotection—against SKLBPA1 infection.

FIG. 9 shows immunoprotection—against CRPA infection.

FIG. 10 shows an experimental protocol of combination of a vaccine and antibiotics against infection.

FIG. 11 shows the experimental results of combination of a vaccine and antibiotics against infection.

DETAILED DESCRIPTION

To make the objective, the technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in combination with drawings. It is obvious that the described embodiments are some of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making inventive effort shall belong to the protection scope of the present invention.

The Pseudomonas aeruginosa vaccine of the present invention comprises (i) irradiation-inactivated Pseudomonas aeruginosa cells and/or (ii) Pseudomonas aeruginosa membrane vesicles. FIG. 1 is a Transmission Electron Microscopy (TEM) photograph of purified membrane vesicles.

Embodiments 1-3 introduce some isolation methods for preparing vesicles The vesicles can be isolated from non-irradiated bacteria or isolated from irradiated bacteria or obtained by other methods.

Embodiment 1 Isolation Method for Bacterial Membrane Vesicles

In some embodiments, a method for isolating membrane vesicles from Pseudomonas aeruginosa comprises the following steps: 1) isolating bacterial cells in bacterial solution for culture of bacteria from culture medium, and collecting supernatant 1; 2) centrifuging the supernatant 1 with a high-speed centrifuge, and collecting supernatant 2; and 3) centrifuging the supernatant 2 with an ultra-high-speed centrifuge to precipitate membrane vesicles.

Further, isolation method in the step 1) comprises centrifugation, column chromatography, or dialysis bag concentration.

Further, the supernatant 2 collected in the step 2) is subjected to dialysis bag concentration prior to the step 3). In some embodiments, the dialysis bag selected can concentrate substances greater than 100 KD.

Further, the membrane vesicles are resuspended with a buffer solution, the buffer solution comprises 50 mM Tris, 5 mM NaCl and 1 mM MgSO₄ calculated as a volume unit of 1 L and has a pH of 7.4.

In some embodiments, the bacterial cells and the membrane vesicles are prepared as a biological composition, and the preparation method comprises: collecting the bacterial cells isolated in the step 1) in the above isolation method for membrane vesicles, and mixing the bacterial cells with the membrane vesicles obtained in the step 3) to form the biological composition.

Further, in the step 1), the supernatant 1 is filtered with a 0.3-0.5 μM filter to remove impurities.

Preferably, the supernatant 1 is filtered with a 0.45 μM filter to remove impurities.

Further, the isolation method in the step 1) is centrifugation, the centrifugation speed is 100-10000 g, and the centrifugation time is 10-60 min.

Preferably, the centrifugation speed in the step 1) is 400-8000 g, and the centrifugation time is 10-30 min.

Further, the high-speed centrifugation speed in the step 2) is 5000-25000 g, and the high-speed centrifugation time is 10-100 min.

Preferably, the high-speed centrifugation speed in the step 2) is 10000-20000 g, and the high-speed centrifugation time is 30-60 min.

Further, the ultra-high-speed centrifugation speed in the step 3) is 5000-150000 g, and the ultra-high-speed centrifugation time is 60-600 min.

Preferably, the ultra-high-speed centrifugation speed in the step 3) is 15000-150000 g, and the ultra-high-speed centrifugation time is 60-180 min.

Embodiment 2 Augmentation and Purification of Bacterial Membrane Vesicles

Further, in some embodiments, the method for preparing bacterial membrane vesicles further comprises the following steps: 1) Augmentation of membrane vesicles: culturing bacteria to logarithmic growth phase; collecting the bacterial cells, resuspending the bacterial cells and then irradiating them with ionizing irradiation to obtain irradiated bacteria; 2) Isolation and purification of membrane vesicles: isolating membrane vesicles produced by the irradiated bacteria from the irradiated bacteria to obtain the membrane vesicles using the method for isolating membrane vesicles described in Embodiment 1.

Further, the ionizing irradiation is X-rays, and the irradiation dose is 500-3000 Gy. The irradiation dose specifically comprises: 500-600 Gy, 600-700 Gy, 700-800 Gy, 800-900 Gy, 900-1000 Gy, 1000-1100 Gy, 1100-1200 Gy, 1200-1300 Gy, 1300-1400 Gy, 1400-1500 Gy, 1500-1600 Gy, 1600-1700 Gy, 1700-1800 Gy, 1800-1900 Gy, 1900-2000 Gy, 2100-2200 Gy, 2200-2300 Gy, 2300-2400 Gy, 2400-2500 Gy, 2500-2600 Gy, 2600-2700 Gy, 2700-2800 Gy, 2800-2900 Gy and 2900-3000 Gy.

Preferably, the irradiation dose is 500-1000 Gy. The irradiation dose specifically comprises: 500-600 Gy, 600-700 Gy, 700-800 Gy, 800-900 Gy and 900-1000 Gy.

Further, OD₆₀₀ value of the bacteria in logarithmic growth phase in the step 1) is 0.3-0.8.

Preferably, the OD₆₀₀ value of the bacteria in logarithmic growth phase in the step 1) is 0.5-0.7.

Further, in the step 1), the bacterial cells are resuspended with phosphate buffer saline or sterile normal saline.

Preferably, in the step 1), the bacterial cells are resuspended with phosphate buffer saline.

Further, in the step 1), the bacterial cells are resuspended to an OD₆₀₀ value of 20-80.

Preferably, in the step 1), the bacterial cells are resuspended to an OD₆₀₀ value of 40-60.

The content of nucleic acids and the content of proteins in the membrane vesicles prepared by the above method are increased by 10-20 times, compared with those prepared from bacteria not irradiated with ionizing irradiation.

The membrane vesicles prepared by the present invention have various application scenarios: for example, (i) the membrane vesicles can be used as an immunogen; (ii) the membrane vesicles can be used as an immune response enhancer; (iii) the membrane vesicles can be used as a vaccine for treating bacterial infectious diseases; (iv) the membrane vesicles can be used as a vaccine adjuvant (in some embodiments, the vaccine adjuvant non-specifically changes or enhances the antigen-specific immune response of the body); (v) the membrane vesicles can be used as an antigen-presenting cell function enhancer.

The above antigen-presenting cell includes dendritic cells (i.e., DC cells), macrophages and B cells. The membrane vesicles obtained by irradiation, isolation and purification can be used as an enhancer for the maturation of the DC cells, and specifically, used as an enhancer for promoting the significant up-regulation of cell surface molecules CD80, CD86 and MHCII molecules of bone marrow-derived dendritic cells.

In some embodiments, the membrane vesicles prepared by the present invention can be combined with DC cells in preparation of a proliferation agent for CD4⁺T cells. Specifically, the method for promoting proliferation of CD4⁺T cells comprises the following steps: co-culturing membrane vesicles-stimulated and OVA-antigen-phagocytosed DCs with CFSE-labeled CD4⁺T lymphocytes in vitro, wherein the membrane vesicles are prepared by irradiation.

Embodiment 3 A Method for Isolating and Preparing Bacterial Membrane Vesicles

In some embodiments, the method for isolating and preparing bacterial membrane vesicles comprises the following steps:

1. Culturing bacteria to logarithmic growth phase, wherein OD₆₀₀ value of the bacteria in logarithmic growth phase is 0.3-0.8, and the OD₆₀₀ value of 0.5-0.8 is preferably selected (fermentation can also be performed here to further enrich bacterial cells); collecting bacterial cells, and resuspending the bacterial cells with an appropriate amount of phosphate buffer solution, wherein the ratio of the amount of the added phosphate buffer solution to the total amount of the bacterial cells is that the OD₆₀₀ value of the amount of the bacteria contained in every 1 ml of solution is 20-80, and the OD₆₀₀ value of 40-60 is preferably selected; after resuspension, irradiating the bacterial cells with ionizing irradiation to obtain irradiated bacteria; preferably, irradiating with X-rays, with an irradiation dose of 500-3000 Gy. The irradiation dose specifically comprises: 500-600 Gy, 600-700 Gy, 700-800 Gy, 800-900 Gy, 900-1000 Gy, 1000-1100 Gy, 1100-1200 Gy, 1200-1300 Gy, 1300-1400 Gy, 1400-1500 Gy, 1500-1600 Gy, 1600-1700 Gy, 1700-1800 Gy, 1800-1900 Gy, 1900-2000 Gy, 2100-2200 Gy, 2200-2300 Gy, 2300-2400 Gy, 2400-2500 Gy, 2500-2600 Gy, 2600-2700 Gy, 2700-2800 Gy, 2800-2900 Gy and 2900-3000 Gy.

2. Collecting bacterial solution, centrifuging the bacterial solution and collecting supernatant, and filtering the supernatant with a 0.3-0.5 μM filter to remove the bacteria; wherein the centrifugation speed is 400-8000 g; and the centrifugation time is 10-30 min.

3. Centrifuging the filtered supernatant with a high-speed centrifuge, collecting supernatant, and removing flagella; wherein the high-speed centrifugation speed is 10000-20000 g; and the high-speed centrifugation time is 30-60 min.

4. Centrifuging the supernatant after removal of the flagella with an ultra-high-speed centrifuge to precipitate membrane vesicles; wherein the ultra-high-speed centrifugation speed is 15000-150000 g; and the ultra-high-speed centrifugation time is 60-180 min.

5. Collecting the membrane vesicles to obtain purified membrane vesicles.

Preparation, isolation and purification of membrane vesicles by irradiating Pseudomonas aeruginosa PAO1 with ionizing irradiation:

1. Streaking Pseudomonas aeruginosa PAO1 recovered from −80° C. onto LB plates, and culturing them in an incubator at 37° C. for 16-18 h.

2. Picking monoclonal colonies from the LB plates, inoculating the monoclonal colonies in 20 mL of LB liquid medium, and culturing them at constant temperature of 37° C. at 250 rpm for 16-18 h.

3. Inoculating overnight bacterial solution into 1 L of LB medium to an initial concentration of 0.05 OD₆₀₀/mL and culturing the bacteria to logarithmic growth phase at 37° C. at 250 rpm, and measuring OD₆₀₀ value of the bacterial solution.

4. Transferring the above bacterial solution of the step 3 to a centrifugal barrel, centrifuging the bacterial solution at 5,000 g for 20 min, collecting the bacterial cells and resuspending the bacterial cells with normal saline, and adjusting the concentration of the bacterial cells to about 50OD.

5. Placing the above bacterial solution in an irradiator with an irradiation dose of 1000 Gy.

6. Centrifuging the irradiated bacterial solution at 8,000×g for 20 min twice and collecting supernatant; filtering the supernatant with a 0.45 μM filter to remove the bacteria and collecting the supernatant again; at the same time, coating a small amount of the supernatant onto the LB plates and culturing them at 37° C. for 24-72 h to confirm that viable bacteria do not exist.

7. Centrifuging the supernatant of the step 6 with a high-speed centrifuge to remove flagella in the supernatant.

8. Centrifuging the supernatant of the step 7 with an ultra-high-speed centrifuge to precipitate membrane vesicles.

9. Discarding the supernatant, resuspending the precipitate with MV buffer, and storing it at −80° C.

10. Observing the extracted membrane vesicles of the normal group and the membrane vesicles of the experimental group of the present invention by transmission electron microscopy. Determining contents of substances in the extracted membrane vesicles of the normal group and the membrane vesicles of the experimental group of the present invention at the same time, including determining the content of DNAs, the content of RNAs and the content of proteins therein. Finally, measuring particle sizes of the extracted membrane vesicles of the normal group and the membrane vesicles of the experimental group of the present invention.

Experimental results:

According to the results of transmission electron microscopy, the ionizing irradiation can stimulate Pseudomonas aeruginosa PAO1 to produce membrane vesicles. The membrane vesicles are shown in FIG. 1 .

Determination results of contents of substances in membrane vesicles: the content of nucleic acids and the content of proteins in the membrane vesicles prepared in the experimental group (i.e., stimulation of ionizing irradiation is conducted) are increased by 10-20 times compared with those in the normal control group (i.e., stimulation of ionizing irradiation is not conducted).

TABLE 1 Determination of contents of substances in membrane vesicles Irradiation DNA RNA Protein Endotoxin dose Gy ng/μL ng/μL μg/mL (EU/ml) — 24.8 19.7 262.7 1.28 × 10⁵ 980 469.0 364.0 4551.0 1.07 × 10⁶

Embodiment 4 Immunomodulatory Effects of Irradiated Bacterial Membrane Vesicles—Promoting Maturation of Dendritic Cells

Dendritic cells (DCs) are the main antigen-presenting cells of the body, and have the main function of phagocytosing and processing antigen molecules as well as presenting them to T cells. The DCs are the known most powerful and the only professional antigen-presenting cell that can activate resting T cells in the body, and are a key link in initiating, regulating and maintaining immune responses. The maturation of the DCs determines the immune response or immune tolerance of the body. Co-stimulatory molecules B7 (B7-1=CD80 and B7-2=CD86) on the surfaces of the DCs can be bound to CD28 or CD152 molecules on the surfaces of T cells, to enhance or weaken the MHC-TCR signal transduction between DCs and T cells. The main characteristics of mature DCs are changes in the expression of co-stimulatory molecules CD80 and CD86, reduced ability to phagocytose antigens and enhanced the ability to process and present antigens (increased MHCII molecules expression), and interaction with T lymphocytes.

1. Culture and induction of mouse bone marrow-derived dendritic cells (BMDC): taking 6-8 week old C57 female mice, aseptically separating mouse femurs, removing the muscles on the femurs, and cutting both ends of the femurs; rinsing the bone lumens with PBS until the bone lumens turn white; filtering PBS suspension and then centrifuging it at 1200 rpm for 5 min; removing supernatant; and adding 5 ml of red blood cell lysis buffer to resuspend the cells. After standing for 15 min, centrifuging the lysis product at 1200 rpm for 5 min, and removing the supernatant; adding 50 ml of 1640 complete medium (20 ng/ml GM-CSF, 10% FBS and 50 mM of 2-mercaptoethanol) to resuspend the cells. After uniform mixing, dividing the cells into 5 petri dishes and culturing them in an incubator. Changing the medium every 2 days and collecting the cells on the 7th day.

2. BMDC stimulation: taking the BMDC cells induced for 7 days, and repeatedly blowing the cells in a 6-well plate to detach adherent cells; collecting the cell suspension, centrifuging it at 1100 rpm for 5 min, removing supernatant, and adding 1 ml of medium to resuspend the cells, and adjusting the cell concentration to 1×10⁶/ml after counting viable cells, and inoculating 2 ml of the cells into a new 6-well plate. Each stimulator will be added respectively and uniformly mixed: whole-cell bacteria, whole-cell bacteria +vesicles, and vesicles at a final concentration of 15 μg/mL (based on protein). Continuing to culture them for 24 hours and adding an equal volume of PBS to the growth control group.

3. Maturation markers detection by flow cytometry: after 24 h, taking out the 6-well plate, repeatedly blowing the cells to detach them, collecting the cell suspension into a Flow Cytometry Tube, centrifuging it at 1500 rpm for 3 min, removing supernatant, adding 1 ml of PBS to continue centrifugation at 1500 rpm for 3 min, then removing the supernatant and repeatedly washing for 3 times. Adding CD11c/CD80/CD86/MHCII antibodies and incubating at room temperature for 30 min in the dark; at the same time, setting an isotype control group as the negative control group (adding isotype controls of CD11c/CD80/CD86/MHCII). After incubation, adding PBS to wash twice, then adding 200 μ1 of PBS to resuspend the cells, and detecting the cells by flow cytometry.

4. Result processing: analyzing the ratio of CD80/CD86/MHCII in CD11c cells by flow cytometry software.

Experimental results: Compared with the whole-cell bacteria, the vesicles of the experimental group (MVs) treated by X-rays can significantly up-regulate the surface costimulatory molecules CD80, CD86 and MHCII of DCs after stimulation, and these surface molecules are markers of dendritic cell maturation. In conclusion, it is proved that the vesicles can significantly promote the differentiation and maturation of DCs.

The phagocytic ability of DC cells is detected by detecting the fluorescence intensity of FITC-dextran: DC cells have strong antigen endocytosis and processing abilities. The DC cells have strong phagocytic ability in an immature state when not in contact with antigen. After in contact with antigen and activated, the DC cells become mature with low phagocytic ability and enhanced antigen-presenting ability. In the experiment, by detecting the fluorescence intensity of the FITC-dextran, the amount of the dextran phagocytosed by DC is determined to detect whether the phagocytic ability of DC is enhanced.

1. Culture and induction of BMDC cells (same as the above).

2. Stimulation: collecting the cells on the 7th day, blowing down all the cells, then centrifuging and resuspending the cells for counting, then inoculating the cells into a 6-well plate with 1×10⁶ cells per well, and respectively adding the stimulator: adding an equal volume of PBS to the GC group, adding the same concentration of membrane vesicles (by protein level) to the control group and the treatment group and then culturing them at 37° C. for 24 h.

3. Phagocytosis and detection: adding the dextran (5 μg/ml), and after culture for 1 h, aspirating the cells into a Flow Cytometry Tube; washing the cells with PBS for 3 times; adding CD11c antibody and incubating at room temperature for 30 min in the dark; washing the cells with PBS for 3 times; and detecting the fluorescence of FITC by flow cytometry.

4. Result processing: analyzing the ratio of FITC in CD11c cells by flow cytometry software.

Experimental results: in order to detect the phagocytic function of DCs, the present invention uses FITC-dextran as a model antigen for phagocytosis of DCs and detects the mean fluorescence intensity value of FITC of CD11c+DCs. The experimental result shows that after the DCs are stimulated by membrane vesicles, the mean fluorescence intensity value of FITC is significantly reduced compared with that of the GC group (growth control group). This experimental result proves again that the vesicles can promote the maturation of DCs, thereby reducing their ability to take up antigen.

Embodiment 5 Interaction Between Mature DCs and T Cells Stimulated by Bacterial Membrane Vesicles in X-ray Treatment Group

A. Interaction between mature DCs and CD4+T cells:

The effective cross-antigen presentation of extracellular proteins by DCs plays an important role in the induction of specific cellular immune responses. Therefore, the cross-presentation effect of OVA antigen by DCs stimulated by membrane vesicles is detected. 72 h after co-culture of DCs-T cells, the proliferation of OT-II CD4⁺T lymphocytes is detected by CFSE flow cytometry. Fluorescent dye CFSE (CFDA-SE), namely carboxyfluorescein diacetate, succinimidyl ester, is a cell staining reagent that can fluorescently label live cells. CFDA-SE can be irreversibly coupled to cellular proteins by binding to intracellular amines after entering cells. In the process of cell division and proliferation, the CFSE-labeled fluorescence can be equally distributed to two daughter cells, and the fluorescence intensity is half that of the parental cells. Therefore, the percentage of cells with weak CFSE fluorescence can be counted by flow cytometry to obtain the proportion of proliferating cells.

1. Culture and induction of BMDC cells (same as the previous embodiment).

2. Antigen phagocytosis: culturing DCs which are cultured for 7 days in a medium containing 10 μg/ml OVA for 24 h to serve as GC (growth control group); adding vesicles to the MVs group, then centrifuging and collecting antigen-phagocytosed DCs; resuspending the DCs in a normal medium; and applying the DCs in a 96-well plate at a density of 2×10⁴ cells/well, with 100 μl per well, and 3 replicate wells per group.

3. T cell extraction: on the second day, isolating and enriching OVA-specific CD4+T lymphocytes from the spleens of OT-II mice by a magnetic negative selection beads kit from Stem Cell Technologies company.

4. Co-culture of DC and T cells: labeling the sorted CD4⁺T cells with 1 μM CFSE according to the kit instructions. After labeling, washing the cells for 3 times with PBS and adding the cells to the 96-well plate at a density of 10⁵ cells/well to a final culture volume of 200 μl (CD4:DC =5:1).

5. On the 3rd day after co-culture, detecting the proliferation of CD4+T cell population by CFSE decrement by flow cytometry.

Experimental results: co-culturing membrane vesicles-stimulated and OVA-antigen-phagocytosed DCs with CFSE-labeled OT-II mouse CD4⁺T lymphocytes in vitro. The analysis results of flow cytometry for CFSE fluorescence intensity show that the proportion of proliferating CD4+T cells is increased. The membrane vesicles (14.05%) can significantly increase the proliferation-promoting effect of OVA-antigen-phagocytosed DCs (6.80%) on specific CD4⁺T cells. See FIG. 2 and FIG. 3 for details.

B. Promotion of T cell proliferation by DCs treated by the membrane vesicles:

The effective cross-antigen presentation of extracellular proteins by DCs plays an important role in the induction of specific cellular immune responses. Therefore, the cross-presentation effect of OVA antigen by DCs stimulated by membrane vesicles is detected. 72 h after co-culture of DCs-T cells, the proliferation of T lymphocytes is detected by CFSE by flow cytometry. Fluorescent dye CFSE (CFDA-SE), namely carboxyfluorescein diacetate, succinimidyl ester, is a cell staining reagent that can fluorescently label live cells. CFDA-SE can be irreversibly coupled to cellular proteins by binding to intracellular amines after entering cells. In the process of cell division and proliferation, the CFSE-labeled fluorescence can be equally distributed to two daughter cells, and the fluorescence intensity is half that of the parental cells. Therefore, the percentage of cells with weak CFSE fluorescence can be counted by flow cytometry to obtain the proportion of proliferating cells.

1. Culture and induction of BMDC cells (same as the previous embodiment).

2. Antigen phagocytosis: culturing DCs which are cultured for 7 days in a medium for 24 h to serve as GC (growth control group); adding vesicles to the MVs group, then centrifuging and collecting antigen-phagocytosed DCs; resuspending the DCs in a normal medium; and applying the DCs in a 96-well plate at a density of 4×10⁴ cells/well, with 100 μl per well, and 3 replicate wells per group.

3. T cell extraction: on the second day, isolating and enriching the T cells of the mice from the spleens of mice one week after one MVs immunization using a magnetic negative selection beads kit from Stem Cell Technologies company.

4. Co-culture of DC and T cells: labeling the sorted T cells with 1 μM CFSE according to the kit instructions. After labeling, washing the cells for 3 times with PBS and adding the cells to the 96-well plate at a density of 4×10⁵ cells/well to a final culture volume of 200 μl (CD3:DC =10:1).

5. On the 3rd day after co-culture, detecting the proliferation of CD3⁺, CD8⁺ and CD4⁺T cell populations by CFSE decrement by flow cytometry.

Experimental results: co-culturing membrane vesicles-stimulated and OVA-antigen-phagocytosed DCs with CFSE-labeled OT-II mouse CD4⁺T lymphocytes in vitro. The analysis results of flow cytometry for CFSE fluorescence intensity show that the proportion of proliferating CD4⁺T cells is increased. As shown in the figure, the fluorescence intensity of the whole-cell bacteria plus vesicle stimulation group is 63.5%, and the fluorescence intensity of the vesicle stimulation group is 71%. It indicates that DCs after vesicle treatment can significantly stimulate the proliferation of CD4⁺T cells. See FIG. 4 .

Embodiment 6 In Vivo Protection Experiment of Pseudomonas aeruginosa Vaccine Against COPD Complicated with Pseudomonas aeruginosa Pulmonary Infection

1. Experimental Groups

The experimental mice (C57BL/6 mice, weight of 17-19 g, female, 60 mice in total) are grouped. Eight groups are set in this experiment, comprising a normal control group (without any experimental treatment), a COPD model group (elastase treatment, and establishment of a COPD model), a COPD +immunization group (elastase treatment, and immunization with different procedures), a COPD +infection group (elastase treatment, establishment of a COPD model, and then infection), and a COPD+infection+immunization group (elastase treatment, establishment of a COPD model, immunization and infection) (see Table 2 for details).

TABLE 2 Experimental Groups Bacteria Number of Group Elastase for animals number Group name treatment Immunization infection (count) 1 Normal control group — — — 4 2 COPD model group + — — 8 3 COPD + immunization group 1 + + — 8 (injection take places at 0, 1st and 2nd weeks) 4 COPD + immunization group 2 + + — 8 (injection take places at 0, 3rd and 7 th days) 5 COPD + infection group + — SKLB 8 6 COPD + infection + immunization + + PA1 8 group 1 (injection take places at 0, 1st and 2nd weeks) 7 COPD + infection + immunization + + 8 group 2 (injection take places at 0, 3rd and 7th days) 8 COPD + infection group + — 8

2. Immunization

The mice are immunized by subcutaneous injection of the vaccine (10⁸ CFU/ml) at underarm, 100 μ/mouse, for 3 times (injection take places at 0, 3rd and 7th days, or at 0, 1st, and 2nd weeks). The mice are infected and challenged with bacteria 1 week (7 days) after the last immunization.

3. Establishment of COPD Model Mice

3 days before the last immunization (i.e., 10 days before infection and challenge), the mice in groups 2-8 are anesthetized with isoflurane, and treated with elastase (Sigma-Aldrich) by airway perfusion (0.44U/25L/mouse) to establish a COPD model. The normal control group (i.e., group 1) is perfused with an equal volume of phosphate buffer saline (PBS).

4. Pulmonary Function and Pathological Examination

On the 10th day after airway perfusion of elastase, the mice in groups 1-5 are anesthetized by intraperitoneal injection of pentobarbital (30 mg/kg), and pulmonary function is tested after tracheal intubation. Subsequently, the mice are sacrificed, and the lung tissue was taken to prepare sections, and HE staining was performed to observe the symptoms and degree of small airway obstruction, cell infiltration, and emphysema.

5. Pseudomonas aeruginosa Infection and Challenge

5.1 Recovery of Pseudomonas aeruginosa

Pseudomonas aeruginosa SKLB PA1 is taken out from an ultra-low temperature freezer and recovered to TSA plates, and cultured overnight at 37° C.

5.2 Overnight Bacteria Culture in a Shaker

Monoclonal colonies are picked from the TSA plates respectively into TSB for shaking culture at 37° C. at 220 rpm overnight.

5.3 Culture Expansion

The overnight cultured bacterial solution obtained from the step 5.2 is taken to measure OD₆₀₀, and is inoculated into 20 ml of TSB (250 ml conical flask); and the bacteria are shaken at 37° C. at 220 rpm to logarithmic growth phase.

5.4 Centrifugation and Washing

The bacterial solution obtained from the step 5.3 is collected into a 50 ml centrifuge tube, and centrifuged at 4100 rpm (3000×g) at room temperature for 10 min; the supernatant is discarded and the precipitate is resuspended in 2 mL of NS (normal saline) (about 5 OD/ml); and the bacterial solution is adjusted to 0.02 OD₆₀₀ (about 5×10⁶ CFU/ml).

5.5 Challenge

One week after the last immunization (i.e., the 7th day after the last immunization, and the 10th day after the airway perfusion of elastase), the mice are anesthetized by intraperitoneal injection of 10% chloral hydrate at 40 μl/10 g body weight, and the airway is exposed by surgical incision of the skin and muscle adjacent to the airway. 50 μl of bacterial solution (1-7×10⁵ CFU) is injected into the airway; and then the wound is sutured.

5.6 Plate Coating and Counting

The adjusted bacterial solution is diluted 10⁴-fold by NS, coated to a TSA plate by a spiral coater, and cultured at 37° C. overnight, and CFU is counted by a colony counter.

6. Viable Bacteria Counting of the Lung Tissue 24 Hours After Infection

The mice in each group are sacrificed by dislocation of cervical vertebrae 24 hours after infection. Lung tissue is taken aseptically, homogenized, coated onto TSA plates, and cultured overnight at 37° C.; and CFU is counted.

7. Data processing: LOG₁₀CFU Scatter Plots of Lung Tissue are Drawn using Graphpad Prism Software; and LOG₁₀CFU is Calculated.

Experimental Results:

1. Pulmonary function test: on the 10th day after airway perfusion of elastase, the mice in groups 1-5 are anesthetized by intraperitoneal injection of pentobarbital (30 mg/kg), and pulmonary function is tested after tracheal intubation. The results are shown in Table 3. The results of pulmonary function test show that after the mice are perfused with elastase at 0.44 U/25L/mouse, the indexes such as offset (sensor drift), MV (minute volume), AV (accumulative volume), VolBal (inspiration-to-expiration ratio), pExp (expiratory pressure difference) are higher or significantly higher than those of normal mice; and the indexes such as dVPEF (%) (ratio of volume at peak tidal expiratory flow to expiratory tidal volume), FIay (flow rate), F (respiratory rate), EF25 (25s expiratory volume) are lower or significantly lower than those of normal mice, indicating that the COPD model is successfully established.

TABLE 3 Partial Pulmonary Function Indexes of COPD Model Mice Normal COPD COPD + control model infection Pulmonary Function group group group Index (n = 3) (n = 5) (n = 6) Offset −1.242 ± −1.109 ± −1.023 ± (sensor drift) 0.052 0.034** 0.028**## MV 22.968 ± 35.410 ± 25.636 ± (minute volume) 10.256 4.484* 6.267# AV 100.399 ± 660.736 ± 2410.808 ± (accumulative volume) 83.874 185.161** 135.771**## VolBal 26.534 ± 46.355 ± 83.293 ± (inspiration-to-expiration 7.190 5.460** 22.238**## ratio) EF25 0.474 ± 0.101 ± 0.049 ± (25 s expiratory volume) 0.214 0.073** 0.029** dVPEF (%) 75.336 ± 60.329 ± 52.869 ± (ratio of volume at peak 9.339 18.577 21.482 tidal expiratory flow to expiratory tidal volume) Flav −0.920 ± −1.227 ± −1.144 ± (flow rate) 0.347 0.194 0.146 pExp −43.862 ± −32.905 ± −10.337 ± (expiratory pressure 0.105 21.792 12.230** difference) F 177.810 ± 175.614 ± 119.576 ± (respiratory rate) 35.444 23.327 12.546*## (Note: *Compared with the normal control group, p < 0.05 represents statistically significant differences; **compared with the normal control group, p < 0.01 represents statistically highly significant differences. #Compared with the COPD model group, p < 0.05 represents statistically significant differences; and ##compared with the COPD model group, p <0.01 represents statistically highly significant differences.)

3. Pathological Examination Results of Tissue

The pathological examination results of lung tissue of the mice are shown in FIG. 5 and FIG. 6 .

In the lung tissue of the mice in the normal control group (FIG. 5 ), a small amount of red blood cells leaked from local alveoli (which is a common phenomenon in sampling operation) and a small amount of inflammatory cell infiltration (lymphocytes) can be seen, and there is no other abnormality.

In the lung tissue of the mice in the COPD model group (FIG. 6 ), it can be seen that a moderate amount of inflammatory cells (mainly lymphocytes) are distributed along the bronchioles and microvessels, that the perivascular interstitial tissue has edema, and that a small amount of red blood cells are leaked from the alveoli. It also can be seen that in some areas, the pulmonary septum is thinned and obviously ruptured, and that pulmonary bullae are formed (pulmonary radiation alveolitis). It indicates that the COPD model is established successfully.

4. Infective dose of Pseudomonas aeruginosa in Mouse Lungs

The concentration of bacterial suspension of Pseudomonas aeruginosa SKLBPA1 used in the experiment is 0.01 OD/ml, and the number of colonies is counted for the remaining bacterial suspension after airway perfusion. The result is that the bacteria solution for infection has a concentration of 4.5×10⁵CFU/ml.

5. Bacterial Load in the Lung Tissue of the Mice 24 h after Pseudomonas aeruginosa Infection.

The mice are sacrificed 24 h after infection, the lung tissue is aseptically taken, and the number of colonies is counted. The mean and standard deviation of each group are compared. The results indicate that the vaccine PA1 has obvious in vivo protective effects on COPD complicated with Pseudomonas aeruginosa pulmonary infection through two immunization procedures of 0, 3, 7d (injection take places at 0, 3rd, and 7th days, upward triangle in FIG. 7 ) and 0, 1, 2 w (injection take places at 0, 1st and 2nd weeks, downward triangle in FIG. 7 ), and the bacterial load of the lung tissue can be reduced by about 2 logs. See FIG. 7 and Table 4.

TABLE 4 Bacterial Load in the Lung Tissue of the COPD Mice 24 h after Pseudomonas Aeruginosa Infection Challenge (Infection) Concentration of Bacterial solution Time after for Infective Immunization COPD Last Bacterial infection Dose Lung Groups Vaccine Procedure Modeling Immunization strain (CFU/ml) (CFU) LOG₁₀CFU 6 PAI 0, 1, 2 w + 7 d SKLBPA1 0.9 × 10⁷ 4.5 × 10⁵   2.77 ± 0.78** 7 0, 3, 7 d + 7 d  2.23 ± 2.06* 8 − + − 4.65 ± 0.35 (Note: *Compared with the model group, p < 0.05 represents statistically significant differences; **compared with the model group, p < 0.01 represents statistically highly significant differences.)

Embodiment 7 Against Multidrug-Resistant Bacterial Infection Experiment (Against CRPA)

Experimental steps: 3 groups are set in the experiment (model group, immunization group SKLB-V1 and aztreonam treatment group). The mice in the immunization group are subcutaneously immunized with SKLB-V1 in the groin at 1×10⁷ CFU/100 μL, with 3 injections take place at 0, 1st and 2nd weeks respectively. One week after the last immunization, SKLBPA1 (which is the homologous strain of vaccine bacteria) and clinical carbapenem-resistant Pseudomonas aeruginosa (CRPA) C58 (from sputum, the drug sensitivity information of clinical C58 is shown in Table 7, which is different from the serotype of vaccine bacteria) are used for challenge. In the aztreonam treatment group, aztreonam of 100 mg/kg/time is administered intraperitoneally at 2, 4, 6 and 8 h after infection, for a total of 4 times. 4 h (i.e., 2 h after the first administration of aztreonam) and 24h after infection, the lung tissue is taken to count the bacterial load (CFU) respectively.

The experimental results are shown in FIG. 8 and FIG. 9 . The bacterial load of the mice in the immunization group is reduced by 2 logs after 24 h, compared with that of the model group, which shows that the vaccine of the present invention is effective in reducing the bacterial load (FIG. 8 and FIG. 9 ). The bacterial load of the mice in the immunization group is reduced by 3 logs after 24 h, compared with that of the aztreonam treatment group, which shows that the vaccine of the present invention is superior to aztreonam (FIG. 8 and FIG. 9 ). The results suggest that the removal efficiency of bacteria exerted by specific immunity established by the vaccine of the present invention in the mice is better than the removal efficiency of bacteria of aztreonam.

TABLE 5 Immunoprotection—against SKLBPA1 Pulmonary Infection Time Vaccine or Bacteria for Bacterial Load Group Name Point Drug Infection log₁₀CFU Model  4 h — SKLBPA1 4.50 ± 1.05 SKLB-V1 SKLB-V1 SKLBPA1 2.41 ± 2.08 Aztreonam Aztreonam SKLBPA1 4.68 ± 0.22 Model 24 h — SKLBPA1 4.69 ± 1.03 SKLB-V1 SKLB-V1 SKLBPA1 2.43 ± 0.94 Aztreonam Aztreonam SKLBPA1 4.31 ± 1.27

TABLE 6 Immunoprotection—against CRPA (Clinically Isolated bacterial strain C58) Pulmonary Infection Time Vaccine Bacteria for Bacterial Load Group Name Point or Drug Infection logwCFU Model  4 h — C58 4.57 ± 0.12 SKLB-VI SKLB-V1 C58  5.40 ± 0.82* Aztreonam Aztreonam C58 6.17 ± 1.55 Model 24 h — C58 3.85 ± 0.86 SKLB-VI SKLB-V1 C58 1.67 ± 0.53 Aztreonam Aztreonam C58 4.69 ± 1.24

TABLE 7 Drug Sensitivity Information of Clinical Isolated bacterial strain C58 Drug Resistance (R) Intermediary (I) Sensitivity (S) ceftazidime gentamicin tobramycin piperacillin amikacin meropenem colistin piperacillin/tazobactam ticarcillin cefepime aztreonam ciprofloxacin levofloxacin ticarcillin/clavulanic acid

Embodiment 8 Efficacy Against Infection of Vaccine Alone or in Combination with Antibiotics

A total of 8 groups are set in the experiment (model group, V1 immunization group, V1 immunization+aztreonam treatment group, and aztreonam treatment group ATM). The mice in the immunization group are subcutaneously immunized with SKLB-V1 in the groin at 1×10⁷ CFU/100 μL, with 3 injections. The immunization procedure of 3 injections can adopt: injection take places at (i) 0, 2nd, and 4th weeks; (ii) 0, 1st, and 2nd weeks; or (iii) 0, 3rd and 7th days. 24 h after the last immunization, the bacteria SKLBPA1 are used for challenge. In the treatment group, aztreonam of 100 mg/kg/time is administered intraperitoneally at 2, 4, 6 and 8 h after infection, for a total of 4 times. 2 h after infection, in the model group (no infection), the lung tissue is taken to count the bacterial load (CFU); 24 h after infection, the lung tissue is taken from each group to count the bacterial load (CFU). A schematic diagram of the above experimental procedures is shown in FIG. 10 .

The experimental results show that the vaccine of the present invention alone or in combination with antibiotics has efficacy against infection (FIG. 11 and Table 8), specifically:

Under the conditions of the model, three immunization procedures 0, 1, 2w (injection take places at 0, 1st, and 2nd weeks), 0, 2, 4w (injection take places at 0, 2nd, and 4th weeks) and 0, 3, 7d (injection take places at 0, 3rd and 7th days) effectively reduce the bacterial load compared with the model group. This indicates that the three immunization procedures can effectively reduce the bacterial load of infection. The effects of the immunization procedures 0, 1, 2w, and 0, 2, 4w are slightly better than that of the immunization procedure 0, 3, 7d, and better than that of aztreonam alone. It is worth noting that the immunization procedure of 0, 3, 7d can also quickly generate an effective immune response, so the vaccine of the present invention can be used to prevent COPD complicated with infection (that is, as a prophylactic vaccine), and can also be possibly used to inhibit infection and alleviate infection after the onset of infection (that is, the possibility of serving as a therapeutic vaccine).

After combining with aztreonam, the efficacy against infection of SKLB-V1 in combination with antibiotic aztreonam in three immunization procedures is significantly better than that of the vaccine alone or the aztreonam alone, which indicates that SKLB-V1 and antibiotic aztreonam have a synergistic effect against infection.

TABLE 8 Efficacy against infection of Vaccine alone or in Combination with Antibiotics Immunization Vaccine Administration Lung Groups Procedure or Drug (Frequency) LOG₁₀CFU V1 (0, 3, 7 d) injection take V1 — 4.66 ± 1.53** places at 0, 3rd, 7th days V1 (0, 1, 2 w) injection take 3.28 ± 1.0** places at 0, 1st, 2nd weeks V1 (0, 2, 4 w) injection take 3.23 ± 0.85** places at 0, 2nd, 4th weeks V1 (0, 3, 7 d) + injection take V1 + 4 times 2.25 ± 1.74** ATM places at 0, ATM (2 h, 4 h, 6 h, 3rd, 7th days 8 h, 100 V1 (0, 1, 2 w) + injection take mg/kg/time) 2.98 ± 1.65** ATM places at 0, 1st, 2nd weeks V1 (0, 2, 4 w) + injection take 2.25 ± 1.42** ATM places at 0, 2nd, 4th weeks ATM — ATM 4 times 4.11 ± 0.44** (2 h, 4 h, 6 h, 8 h, 100 mg/kg/time) Model 2 h — — — 5.77 ± 0.40** Model 24 h — — — 6.86 ± 0.45

The embodiments of the present invention are described above in combination with drawings, but the present invention is not limited to the aforementioned specific embodiments. The aforementioned embodiments are merely illustrative and not limiting. For those of ordinary skill in the art, many forms can be made under the teaching of present invention without departing from the spirit of the present invention and the scope of the claims, all of which shall fall within the protection scope of the present invention. 

1. Use of a Pseudomonas aeruginosa vaccine in the manufacture of a medicament for the prevention and treatment of respiratory system disease, wherein the respiratory system disease is chronic pulmonary insufficiency complicated with bacterial infection, the chronic pulmonary insufficiency is chronic obstructive pulmonary disease and the bacterial infection is Pseudomonas aeruginosa infection; wherin the Pseudomonas aeruginosa vaccine comprises inactivated Pseudomonas aeruginosa and Pseudomonas aeruginosa member visicles, the inactivated Pseudomonas aeruginosa is inactivated by irradiation, and the Pseudomonas aeruginosa member vesicles are isolated from the Pseudomonas aeruginosa inactivated by irradiation.
 2. The use according to claim 1, wherein the respiratory system disease comprises primary respiratory system disease and secondary respiratory system disease.
 3. (canceled)
 4. (canceled)
 5. The use according to claim 14, wherein the chronic obstructive pulmonary disease comprises chronic bronchitis and emphysema.
 6. The use according to claim 14, wherein the chronic obstructive pulmonary disease is acute exacerbation of chronic obstructive pulmonary disease.
 7. (canceled)
 8. (canceled)
 9. The use according to claim 1, wherein immunization procedure of the Pseudomonas aeruginosa vaccine comprises: injection take places at (i) 0, 3rd, and 7th days; (ii) 0, 1st, and 2nd weeks; and (iii) 0, 2nd, and 4th weeks.
 10. (canceled)
 11. (canceled)
 12. The use according to claim 1, wherein the Pseudomonas aeruginosa vaccine prevents Pseudomonas aeruginosa infection, and reduces bacterial load in the respiratory system disease.
 13. The use according to claim 1, wherein content of whole-cell Pseudomonas aeruginosa in the Pseudomonas aeruginosa vaccine comprises: 1×10⁴-1×10¹¹/injection.
 14. The use according to claim 13, wherein the content of the whole-cell Pseudomonas aeruginosa in the Pseudomonas aeruginosa vaccine comprises: 1×10⁴/injection, 1×10⁵/injection, 1×10⁶/injection, 1×10⁷/injection, 1×10⁸/injection, 1×10⁹/injection and 1×10¹¹/injection.
 15. The use according to claim 1, wherein the Pseudomonas aeruginosa vaccine further contains an immunoadjuvant.
 16. The use according to claim 15, wherein the immunoadjuvant is aluminum hydroxide.
 17. The use according to claim 1, wherein administration site of the Pseudomonas aeruginosa vaccine is subcutaneous, muscle and/or mucosa.
 18. The use according to claim 1, wherein the medicament can also contain any pharmaceutically acceptable carrier and/or adjuvant.
 19. The use according to claim 18, wherein the carrier is a liposome.
 20. A pharmaceutical composition for treating respiratory system disease, wherein comprising a Pseudomonas aeruginosa vaccine and at least one antibiotic, wherein the at least one antibiotic is aztreonam, the Pseudomonas aeruginosa vaccine comprises inactivated Pseudomonas aeruginosa and/or Pseudomonas aeruginosa membrane vesicles; wherein the inactivated Pseudomonas aeruginosa is inactivated by irradiation, and the Pseudomonas aeruginosa membrane vesicles are isolated from the Pseudomonas aeruginosa inactivated by irradiation.
 21. (canceled)
 22. (canceled) 